The present invention relates to the field of cancerology. More particularly, the subject of the present invention is a method for the in vitro diagnosis of primary bronchopulmonary carcinoma in a human patient by determination of the presence, beyond a predetermined threshold, of a major transcript of the KLK8 gene of kallikrein 8 in a biological sample derived from that patient.
Primary bronchopulmonary carcinoma is the main cause of death by cancer in man, and this in all the developed countries. Recent data show a clear increase in its incidence in women. The number of new cases per year is estimated at 25,000 in France and at more than 160,000 in the United States, resulting in the death of about 22,000 individuals per year in France and 155,000 in the United States. Worldwide, broncho-pulmonary carcinoma is thought to be responsible for about 900,000 deaths each year, which would correspond to about 18% of the deaths due to cancer. The main etiology of bronchopulmonary carcinoma is tobacco addiction. About 90% of bronchial cancers in men and about 50% in women are attributable to tobacco. Other environmental or occupational factors can also be recognized in bronchial carcinogenesis.
The World Health Organization (WHO) distinguishes between small cell bronchial carcinomas (SCBC), which represent about 20% of cases, and non-small cell bronchial carcinomas (NSCBC) which inter cilia include epidermoid carcinomas, adenocarcinoma, and large cell carcinomas, and which represent about 80% of cases. The epidermoid carcinomas and adenocarcinomas are the most widespread carcinomas.
At the present time, the diagnosis of bronchopulmonary carcinoma is essentially made by pulmonary radiography and thoracic scanning. Bronchial endoscopy making it possible to perform biopsies then confirms the diagnosis. Unfortunately, the indicative symptoms are delayed and not very specific, and the diagnosis is reached at a late stage, thus greatly reducing the efficacy and the feasibility of the existing treatments. In addition, this type of diagnosis requires sophisticated equipment and qualified personnel which is expensive.
Various treatment methods are currently available: surgery, chemotherapy and radiotherapy. These treatments can be carried out either in isolation or consecutively or in combination.
The survival rate for lung cancer is very dependent on the degree of dissemination of the tumor at the time of diagnosis. The overall survival rate at 5 years is of the order of 15%. However, this rate masks substantial disparities. The survival rate of patients having a carcinoma with remote metastasis at the time of diagnosis is less than 5% whereas patients whose “non-small cell carcinoma” (NSCBC) is localized at the time of its discovery exhibit survival rates close to 50%1. These latter are essentially treated by surgical resection of the tumor, an approach which for the time being represents the only curative solution for this type of carcinoma. However, fewer than one patient in 3 can receive such treatment and one patient in 2 treated surgically dies in the months following the operation, following a tumor relapse. Recent progress in the field of the modern chemotherapy of NSCBC makes it possible to envisage adjuvant or neo-adjuvant treatments improving the life expectation of the patients who have undergone surgery2. However, such treatments are not trivial, with an associated mortality rate which is not negligible. In this context, it is important to be able to identify the operable patients exhibiting a high risk of death due to relapse, in order to facilitate the decision whether or not to give neo-adjuvant or adjuvant chemotherapy.
Markers which make it possible to distinguish tumor cells from healthy cells have been sought and studied for years for all carcinomas and in particular broncho-pulmonary carcinoma. They would make it possible to diagnose the disease at an early stage, to establish its prognosis and sensitivity to treatment, and to monitor its progression. In recent years, more than 100 candidates have been suggested as molecular markers for diagnosis of bronchopulmonary carcinoma. The studies have thus envisaged the diagnostic roles of proto-oncogenes, factors involved in the cell cycle, apoptosis or angiogenesis. However, it has been possible to obtain few correlations between the results obtained by different techniques and validations between various cohorts of patients depending on the technique used (immunohistochemistry, immunological assay, DNA chips utilizing various algorithms) and the great diversity of the tumors (histological type, stage, degree of differentiation).
Other markers, belonging to a subfamily of serine proteases, the kallikreins, of which there are 15, have also been tested. Thus in a study using DNA chips, the hKLK11 gene was identified as a marker of endocrine adenocarcinomas of the C2 type3. A similar study has shown that the hKLK5 and hKLK10 genes are overexpressed in epidermoid carcinomas4. Similarly, it has been shown that the hKLK5 and hKLK7 genes, respectively encoding the proteins hK5 and hK7, were overexpressed in the tumor tissues of epidermoid carcinomas, while underexpression of the hKLK7 gene in the tumor tissues is most often observed in patients exhibiting an adenocarcinoma5. However, it has not been possible to establish any link between the differential expression of these hKLK genes and a survival prognosis for patients suffering from bronchopulmonary carcinoma.
The genes of the 15 kallikreins exhibit characteristics in common, among them the presence of several transcripts for the same gene. The transcripts of these genes have also been studied as markers. Thus, it has been shown that three alternative transcripts of the hKLK46 and hKLK57 genes, and one transcript of the hKLK77 gene were overexpressed in the tumors and/or in ovarian cell lines in comparison with non-cancerous tissue.
It is known that the expression profile of the hKLK8 gene gives rise to at least 4 different transcripts, called NT1 to NT4. The transcript NT1 or “neuropsin type 1”, identified by Yoshida S. et al8, encodes a preproenzyme of 260 amino acids containing a secretion signal peptide of 28 amino acids and a very short prosegment of 4 residues which has to be cleaved off to liberate the active form of kallikrein 8. NT1 is regarded as the regular expression form of the gene. The transcript NT2 or “neuropsin-T2”, identified by Mitsui S. et al9, is differentiated from the NT1 form by the insertion of a sequence encoding 45 supplementary amino acids in the carboxy terminal region of the signal peptide. The transcripts NT3 and NT4 were identified by Magklara A. et al10 and encode proteins containing respectively 119 and 32 residues. The protein form predicted from NT3 only possesses one part of the signal peptide of kallikrein 8 and does not conserve the cleavage zone of the latter. The protein predicted from NT4 is made up of the first 23 residues of the signal peptide of kallikrein 8 and of 9 supplementary residues with no identity with kallikrein 8. Magklara A. et al10 have shown that, although the regular expression form of the KLK8 gene, NT1, may be of prognostic value in the context of carcinoma of the ovary, the forms NT3 and NT4 are of no value.